Interferons ("IFN") are a group of mostly glycosylated polypeptides having molecular weights from 10,000 to 40,000. They are produced by vertebrate cells upon exposure to an IFN inducer, such as a virus, a double-stranded RNA, intracellular microbes, microbial products, or various chemical agents (1). IFNs are usually not found in normal healthy cells. They assist the healthy cells of the vertebrate in their defence against viral infections and other attacks. IFNs have been found to have immunomodulator activities.
The nomenclature for interferon polypeptides has not yet been clearly established. According to recent recommendations (2), the classification will be based on the animal of origin (e.g. "Hu" for human origin), the antigenic specificity (types .alpha., .beta., .gamma. etc., based on their antigen-antibody reactions with .alpha.-, .beta.- or .gamma.-IFN-antibodies, respectively), structural and physiological differences (subtypes are indicated by Arabic numbers, e.g. .alpha..sub.1, .alpha..sub.2 etc.) and the type of cells of origin (Le derived from leukocytes, Ly derived from lymphoblastoid cells, F derived from fibroblasts, etc.). Thus, e.g. HuIFN-.alpha..sub.1 (Ly) or HuLyIFN-.alpha..sub.1 indicates an .alpha.-type interferon of subtype 1, which is derived from human lymphoblastoid cells. In addition to these denominations it may become necessary to specify whether the interferon is obtained directly from its progenitor cell or by synthesis in a microorganism, and whether the interferon is a particular subtype or a mixture of two or more interferon subtypes.
The nomenclature concerning the DNA sequences coding for IFN polypeptides, the recombinant DNA molecules and the hosts containing them has not been clearly established either. The nomenclature used for these DNA sequences reflects the interferon which they encode in abbreviated form. E.g., Escherichia coli HB 101 (Z-pBR 322 (Pst)/HcIF-2h) indicates the bacterial strain (E. coli HB 101) containing the recombinant plasmid DNA Z-pBR 322 (Pst)/HcIF-2h, i.e. the plasmid pBR 322 containing at the Pst I site (site of insertion of the foreign DNA) a HcIF-.alpha..sub.1 insert, originated in Zurich (Z). The letter "H" indicates the human origin, "c" indicates a complementary DNA and .alpha..sub.1 stands for the subtype (cf. C. Weissmann, (3)). In the given nomenclature example, the source of the IFN genes (leukocytes) is not indicated. In the present application a similar nomenclature is adopted, whereby however, the source of the IFN genes (lymphoblastoid cells) is indicated.
Three classes of the HuIFNs have been identified up to now: HuIFN-.alpha., HuIFN-.beta. and HuIFN-.gamma.. HuIFN-.alpha. (formerly named LeIFN, leukocyte interferon, or LyIFN, lymphoblastoid interferon) is produced by human leukocytes(fresh cells obtained from the blood of human donors) and by lymphoblastoid cells upon induction, e.g. with a virus (E. A. Havell et al. (4) and A. D. Sagar et al. (5)). It is stable at pH2 (IFNs stable to acid were formerly indicated as "type I") and consists of a mixture of individual interferon polypeptides, which differ mainly in their degree of glycosylation (e.g. M. Rubinstein et al., (6)) and amino acid composition (see below). Of the two components isolated and purified so far, the one with a molecular weight of 15,000 to 18,000 is not glycosylated, whereas the other one with a molecular weight of 21,000 to 22,000 is glycosylated. W. E. Stewart, II et al. (7) have reported that the nonglycosylated interferon has retained most or all of its HuIFN-.alpha. activity. Parts of the amino acid sequence of HuIFN-.alpha. from lymphoblastoid cells have also been reported (K. C. Zoon et al., (8)). Various forms of HuIFN-.alpha. differing structurally and physiologically are already known. Particular human individuals may produce allelic variations of HuIFN-.alpha..
HuIFN-.beta. (formerly FIFN or FiIFN, "type I") is produced by human fibroblasts (e.g. cells of the foreskin of newborns) upon induction with a ds RNA and, to a minor extent, together with HuIFN-.alpha., by human lymphoblastoid cells upon induction with a virus. HuIFN-.beta. is also stable at pH2 (therefore it belongs to "type I"). At least two types of HuIFN-.beta. have been described so far (33, 48). The molecular weights are about 20,000 and 22,000. The amino acid sequence is known in part.
HuIFN-.gamma. [formerly named IIFN (immune interferon or "type II" interferon)] is produced by T-lymphocytes in response to antigens or mitogens. It is acid labile at pH2 and serologically distinct from HuIFN-.alpha. and HuIFN-.beta..
HuIFNs are useful as antivirus, antitumor and anticancer agents. As antiviral agents they can be used to treat e.g. viral respiratory infections, herpes simplex keratitis, acute hemorrhagic conjunctivitis, varicella zoster, hepatitis B, cytomegalic inclusion disease and others.
As antitumor or anticancer agents HuIFNs can be used to treat e.g. osteosarcoma, acute myeloid leukemia, multiple myeloma, Hodgkin's disease, melanoma, breast carcinoma, lymphosarcoma and papilloma and others.
HuIFNs may be used in the form of pharmaceutical preparations for oral or parenteral administration, e.g. pharmaceutical preparations for topical, intravenous, intramuscular, intranasal, intradermal or subcutaneous administration, for example tablets, vials, syrups, solutions or suspensions for oral administration, powders, injection or infusion solutions or suspensions, eye drops, ointments, sprays and the like.
The preparations are usually administered e.g. intramuscularly, once to three times daily in dosages of about 10.sup.6 to about 10.sup.7 units, the treatment depending on the disease, the mode of application and the condition of the patient. Virus infections are usually treated daily or up to three times daily over several days to several weeks, whereas tumors and cancers are treated for several months or years, either one to several times daily or twice or more times weekly.
Up to now HuIFN-.alpha. has been produced only in insufficient quantities through induced human cells, e.g. human lymphoblastoid cells (e.g. from Burkitt's lymphoma "Namalwa" cells) or human leukocytes obtained from the fresh blood of donors. HuIFN-.beta. is obtained mainly from human fibroblasts. It is reported that only 2.6.times.10.sup.9 IU of crude HuIFN-.alpha. are obtained from 800 1 of cultured Namalwa cells, and that only about 10.sup.11 IU of crude HuIFN-.alpha. may be obtained annually at very large blood centers, e.g. the Finnish Red Cross Center in Helsinki. The specific activity of HuIFN-.alpha. is in the order of about 4.times.10.sup.8 to 10.sup.9 IU/mg. The amount of HuIFN-.alpha. required for widespread and commercial application would be very low compared with other pharmaceutical compounds.
One hundred grams of pure HuIFN-.alpha. would provide between 10 and 30 million doses. However, such amounts cannot be produced industrially at a reasonable cost and expense using the human tissue culture and the human leukocyte technology.
Another disadvantage of these large scale production methods is that only mixtures of interferons are obtained and it is cumbersome and expensive to separate them into individual subtypes. Therefore, it has been unsatisfactory to assess the therapeutic applications of pure, individual interferon species so far.
The industrial application of these methods is furthermore limited to HuIFNs produced by human cells which can be cultured (such as human tumor cells and certain fibroblast cells) or to human cells which are available in relatively large amounts (such as leukocytes and lymphocytes). However, all of these methods are expensive and involved.
It was recognized that the solution to the problem of the industrial synthesis of large amounts of individual species of interferons could come from advances in molecular biology, through which it became possible to express a specific non-bacterial eukaryotic gene in bacterial cells. Recently, S. N. Cohen and H. W. Boyer (9) described a general method for replicating biologically functional DNA sequences. This method comprises the steps of cleaving a circular plasmid DNA to give a first linear DNA segment; inserting into this first segment a second linear DNA segment having a gene for a phenotypical trait to give a recombinant DNA molecule (a modified circular plasmid); transforming a unicellular microorganism with this recombinant DNA molecule; growing the transformants together with the non-transformed microorganisms under appropriate nutrient conditions; and isolating the transformants from the parent unicellular microorganisms. The transformants may than produce the desired protein.
The problem of producing a linear DNA sequence coding for interferons and therefrom a recombinant DNA was not solved by Cohen and Boyer.